1. Field of the Invention
This invention relates to a method for the determination of leukocytes in samples of whole blood. More particularly, this invention relates to a method of distinguishing nucleated red blood cells from leukocytes, thereby enabling more accurate complete blood counts.
2. Discussion of the Art
One of the most important clinical results produced by an automated hematology analyzer is the concentration of white blood cells. Nucleated red blood cells tend to interfere with white blood cells, thereby causing the analyzer to either flag the white blood cell counting, thereby rendering the concentration of white blood cells not reportable, or including the nucleated red blood cells in the white blood cell count, thereby producing an inaccurate result. Specimens containing fragile white blood cells or specimens containing hypotonically resistant red blood cells present a problem for automated hematology analyzers. Fragile white blood cells can be lysed along with red blood cells, thereby rendering the white blood cell count inaccurate as being too low. Hypotonically resistant red blood cells, which resist lysing, can be counted as white blood cells, thereby rendering the white blood cell count inaccurate as being too high. Hematology analyzers invalidate these inaccurate results, with the result that the operator is forced to acquire a reportable result by manual means.
Red blood cells are produced from bone marrow progenitors through a programmed series of intermediate developmental stages. All of the precursors of red blood cells are nucleated and are normally located within the bone marrow. As these precursors mature toward the erythrocyte stage, there are progressive decreases in RNA/DNA synthesis and an increase in hemoglobin content. Although nucleated red blood cells may occur as rare events in the blood of normal adults, their frequency is so low that, when seen, they are regarded as a significant abnormality.
The presence of nucleated red blood cells in the blood usually provides valuable insights into the cause of a variety of hematological disorders. When nucleated red blood cells are present in a blood sample, there is a need to ensure that they do not interfere with the white blood cell counting.
Interference of white blood cell counting by nucleated red blood cells generally adversely affect the accuracy of the method and, consequently, the performance of a hematology analyzer for the complete blood count. Historically, this adverse effect has required morphological assessment of the nucleated red blood cell count, along with subsequent correction of the reported leukocyte counts. More recently, however, application of fluorescence flow cytometry has resulted in the development of semi-automated methods and fully automated nucleated red blood cell counting performed as part of the complete blood count.
Manual nucleated red blood cell counting remains the reference method. Manual nucleated red blood cell counting calls for the use of 200 or 100 leukocyte differentials. Despite the fact that accuracy of nucleated red blood cell recognition is high, it is clear that the statistical limitations of the 100 or 200 leukocyte differentials result in apparent inaccuracy and insensitivity when the ratio of nucleated red blood cells to white blood cells is below about 10%. While fluorescence flow cytometry techniques can accurately resolve nucleated red blood cells from other cellular components of the blood, in practice, the expense of these procedures as well as their reliance on many manual intervention steps, prevent widespread application for routine clinical uses.
The nucleated red blood cell count is usually calculated from the ratio of nucleated red blood cell count to the total white blood cell count, or mean nucleated red blood cell count per 100 white blood cells. In a whole blood sample, when nucleated red blood cells are present, there are often other blood components, such as hypotonically resistant red blood cells, platelet clumps, and cell debris to interfere with the nucleated red blood cell count. Those interfering substances are often critical factors, or limitations, of the method to determine the performance or quality of nucleated red blood cell count and white blood cell count for the complete blood count (CBC).
Several automated hematology systems offer nucleated red blood cell estimation as an integral part of the complete blood count. Many automated hematology systems comprise a flow cytometer that has been specifically designed for complete blood count in addition to performing some automated fluorescence flow cytometry techniques. The systems are capable of simultaneously performing a leukocyte differential and nucleated red blood cell analysis. However, some samples from newborn babies, sickle, and thalassemic red blood cells and nucleated red blood cells are resistant to the lytic reagents used during analysis of nucleated red blood cells. These samples often give an incorrect nucleated red blood cell count or an incorrect white blood cell count or both an incorrect nucleated red blood cell count and an incorrect white blood cell count. In order to solve this problem, an extra step is needed to prolong incubation time for lysing most of the hypotonically resistant red blood cells and nucleated red blood cells. However, during any lysing process, it is possible to lyse some of the small or fragile lymphocytes, which may then be misclassified as nucleated red blood cells. In addition, fluorescent dyes and special reagents for performing some automated fluorescence flow cytometry techniques are costly.
Enumeration of nucleated red blood cells is important because nucleated red blood cells interfere with the white blood cell count. Interference with white blood cell counting is a serious problem because users do not have another way to count white blood cells. When instruments invalidate the white blood cells, users need to use another instrument to determine the white blood cell count. Before the advent of automated hematology analyzers, a manual count performed by viewing a grid on a slide was the only way to determine a white blood cell count.
It would be desirable to develop a cost effective, simple, and reliable method for determining nucleated red blood cells, especially for samples that contain hypotonically resistant red blood cells, which frequently contain nucleated red blood cells.